Mitochondrial

• Powerhouses of the cell → most of energy captured

takes place inside it
• Outer membrane → permeable to most metabolites,
contain various enzym (acyl Co-A synthetase,
glycerolphosphate acyltransferase )
• Inner membrane → selectively permeable
• Matrix → contain phospholipid cardiolipin together
with enzymes of resp chain
• Intermembrane space has similar composition
with cytoplasmic and contain adenylyl
kinase and creatine kinase
 A B MATRIX
F1 subunits
OUTER F0 subunits
Phosphorylating MEMBRANE
B complexes
INNER
MEMBRANE

MATRIX

Sonication
Cristae

INNER
MEMBRANE
OUTER
MEMBRANE
Submitochondrial particel
Formed from fragments of the
inner membrance
 Structure and Properties of Mitochondria
Mitochondria are oval-shaped eukaryotic organelles, typically about 2 x 0.5 mm.
They contain the respiratory assembly, the enzymes of the TCA, and the enzymes
of fatty acid oxidation.

From the outside:

cytosol: glycolysis occurs here.
Outer membrane: contains porin (a large
transmembrane protein with a large pore) →
permeable to most small molecules and ions.
Intermembrane space: H+ are pumped here
from the matrix during e- transfers.

Inner membrane: contains all the electron-driven proton pumps → oxidative

phosphorylation takes place here. impermeable to nearly all ions and polar molecules.
Matrix: most of the reactions of the TCA and fatty acid oxidation occur here.
 Inner Mitochondrial Membrane

Cytosolic Side Matrix Side

P Side N Side
Positively charged Inner Membrane Negatively charged
Electron Transfer Reactions in Mitochondria

NADH and FADH2 formed in glycolysis, fatty acid oxidation, and TCA are
energy-rich molecules with a pair of electrons having a high transfer potential.

Oxidative phosphorylation
is the process in which ATP
is formed as a result of the
transfer of electrons from
NADH or FADH2 to O2 by a
series of electron carriers.

Major source of energy in

aerobic organisms!

Of the 30 ATP formed when glucose is completely oxidized to CO2 and H2O,
26 come from oxidative phosphorylation.
 Respiratory chain
• Enzyme complexes in mitochondria → collects and
transports reducing equivalents → directing them to
final reaction with oxygen → form water and ATP
• Reducing equivalents flow through from redox
potential negative to positive
• There are 4 enzyme complexes:
- NADH-Q dehydrogenase or oxydoreduktase/ I
- Succinate-Q dehydrogenase / II
- Cytochromes dehydrogenase / III
- Cytochrome oxidase / IV
 Electron Carriers in the Respiratory Chain
The electrons of NADH enter the chain at NADH-Q reductase.
Complex I: NADH to ubiquinone. Also called
NADH:ubiquinone oxidoreductase. H+ pump.
Complex II: Succinate to ubiquinone. Succinate
dehydrogenase, the only membrane-bound enzyme of
TCA.
Complex III: Ubiquinone to cytochrome c. Also
called cytochrome bc1 complex or
ubiquinone:cytochrome c oxidoreductase. H+ pump.
Complex IV: Cytochrome c to O2. Also called
cytochrome oxidase. H+ pump.
 Complex I: NADH-Q Oxidoreductase

Large enzyme (~880 kDa) consisting of at least 34 polypeptide chains

Proton pump
Contains flavin adenine nucleotide (FMN) and a series of Fe-S clusters of
two kinds: [2Fe-2S] and [4Fe-4S]

The initial step is the binding of NADH and

the transfer of its two high potential e- to the
FMN prosthetic group to give the reduced
form FMNH2:
NADH + H+ + FMN FMNH2 + NAD+
 Complex I: NADH-Q Oxidoreductase
FMN (like FAD) can accept either 1 or 2 e-.
The FMN cofactor of NADH:ubiquinone oxidoreductase accepts 2 e– from NADH
and donates 1 e– at a time to a series of iron-sulfur centers (Fe-S), the second type
of prosthetic group in complex I.

FMN:

Fe-S clusters: Fe
The enzyme contains both [2Fe-2S]
and [4Fe-4S] clusters. Iron atoms
Cys sulfur in these Fe-S complexes cycle
S between Fe2+ (reduced) and Fe+3
(oxidized) states.
 Complex I: NADH-Q Oxidoreductase
e- in the Fe-S clusters are then shuttled to coenzyme Q, also called ubiquinone
(because it is ubiquitous in biological systems).

Ubiquinone (Q) is reduced to a radical semiquinone anion (Q •-) by the uptake of

1 e-. Reduction of this enzyme-bound intermediate by a second e- yields ubiquiniol
(QH2).

The flow of electrons from NADH to QH2

through complex I leads to the pumping of 4
H+ from the matrix to the cytosolic side of the
inner mitochondrial membrane.
 Complex I: NADH-Q Oxidoreductase
The mechanism of conversion of the electron potential into proton-motive force
is not understood yet.

Q is hydrophobic, due to the

its long isoprenoid tail and can
diffuse within the hydrophobic
core of the lipid bilayer

1. NADH binds on the matrix side and transfers its e- to FMN.

2. e- flow to three [4Fe-4S] units centers and then to a tightly bound Q.
3. The pair of e- on bound QH2 are transferred to a [2Fe-2S] center and finally to
a mobile Q in the hydrophobic core of the membrane.
4. Ubiquinol (QH2) diffuses in the mitochondrial inner membrane from
Complex I to Complex III.
Complex II: Succinate Dehydrogenase

In TCA, FADH2 is formed in the

oxidation of succinate to fumarate
catalyzed by succinate dehydrogenase.

This enzyme is an integral membrane

protein of the inner mitochondrial
membrane.
Contains both covalently bound FAD and
Fe-S centers.

e- are transferred from FADH2 to the Fe-

S centers and then finally to Q, which is
reduced to QH2.
 Complex II: Succinate Dehydrogenase

Q is also the port of entry for e- coming directly from:

1. glycolysis (reduction of dihydroxyacetone
phosphate to glycerol-3-phosphate) by glycerol 3-
P-dehydrogenase and the breakdown of
triacylglycerols.
2. oxidation of fatty acids (b oxidation of fatty acyl–
CoA by acyl-CoA dehydrogenase).

Both glycerol-3-phosphate dehydrogenase and acyl-

CoA dehydrogenase are flavoproteins containing FAD
as cofactor.

Succinate dehydrogenase and the other enzymes that transfer e- from FADH2 to
Q, in contrast to NADH-Q reductase, are not proton pumps because the free-
energy change (Go’) of the catalyzed reaction is too small.
Less ATP is formed from the oxidation of FADH2 than from NADH.
Complex III: Ubiquinone:Cytochrome c Reductase
Couples the transfer of e- from ubiquinol (QH2) to cytochrome c with the vectorial
transfer of H+ from the matrix to the intermembrane space.
Cytochrome reductase contains three kinds of cytochromes (cyt b-560, cyt b-566,
and cyt c1) and a [2Fe-2S] center.
A cytochrome is an e- transferring protein that contains a heme prosthetic group.
Cytochrome heme groups in Complex III are of two kinds:
Heme C in cyt c and c1. It
Iron-protoporphyrin IX in is covalently linked to 2 Cys
cyt b. Same as in Mb or Hb residue of the protein.
 Ubiquinone:Cytochrome c Reductase
Ubiquinol transfers one of its two e- to the Fe-S protein in the reductase, which
shuttles this e- to the cyt c1 component of the reductase and then finally to cyt c,
which carries it away from Complex III to Complex IV.

In this process, 2 H+ are pumped to the P side (cytosolic side) of the inner membrane.

This 1 e- transfer converts QH2 to the

semiquinone anion (Q•-).
 Ubiquinone:Cytochrome c Reductase
Cytochrome b (one protein component of Complex III) with its two heme groups
works as a recycling device that enables both e- of QH2 to be used effectively and
transferred to cyt c.
A second Q molecule (bound to cytochrome b)
accepts one e- from the heme groups to form Q•-.

Overall process: one QH2 is oxidized to Q by

donating 1 e- to cyt c and 1 e- to bound Q to
form Q•-.

The second e- is temporarily stored

on bound Q, not to be wasted.
 Ubiquinone:Cytochrome c Reductase
A second QH2 diffuses in the membrane and donates its 2 e- to cyt c and cyt
b, which now has one Q•- bound (where 1 e- from the first transfer was
temporarily stored) that gets reduced to QH2.

Net stoichiometry: 1 QH2 is oxidized to give 1 Q, 2 cytochrome c are reduced

and leave Complex III to take the electrons to Complex IV, and one bound Q is
reduced to QH2.
The Q cycle allows to transfer e- from ubiquinol (which carries 2 e-) coming
from Complex I and II to cytochrome c (which carries 1 e-) that will take them
to Complex IV.
 Proton Motive Force
The flow of e- from NADH or FADH2 to O2 through protein complexes in the
inner membrane of mitochondria leads to pumping of protons (H+) out of the
mitochondrial matrix.

A Proton Motive Force (PMF)

2H+ 4H+
consisting of a pH gradient and a
transmembrane electric potential
is generated.

ATP is synthesized when H+’s

flow back to the mitochondrial
matrix through an enzyme
complex called ATP synthase.
 Coupling of Oxidation and Phosphorylation

Oxidation and phosphorylation are coupled by a proton gradient across the inner
mitochondrial membrane.

electron-motive force Inner membrane space

NADH-Q reductase
proton pumps
cytochrome reductase
cytochrome oxidase

proton-motive force

ATP synthase
Transmembrane complexes containing
multiple redox centers:
phosphoryl potential flavins, quinones, iron-sulfur clusters,
hemes, and copper ions.
 Oxidative phosphorylation
• Oxidative reaction coupled by phosphorylation to
the generation of high energy intermediate (ATP
or other high phosphagen)
• Oxidative phosphorylation at resp chain level →
via NADH form 3 mol ATP and via FADH2 /
FMNH2 form 2 mol ATP
• Phosphorylations at the substrate level →
captured smaller energy
a) High energy phosphates are captured in
kreb’s cycle during the conversion of succinyl
Co-A to succinate.
b) Glycolytic reactions on cytoplasmic:
convertion of 1,3 biphosfoglycerate to 3
phosfoglycerate
 Mechanism of oxidative
phosphorylation

• Mitchell’s chemiosmotic theory:

- energy from oxidation in resp chain →
translocation of H+ (protons) →
electrochemical potential difference in matrix
and intermembrane space → drive the
mechanism of responsible for the formation of
ATP (ATP synthase)
 Mechanism of oxidative
phosphorylation
• Complexes I, III and IV of resp chain is a proton
pump
• Pi + ADP → ATP, by ATP synthase
• ATP synthase is a complex enzyme → consist of
several protein subunits (F1), which attached to
membrane protein complex (F0)
• F1 → project into matrix and contain the
phosphorylation mechanism
F0 → spans the membrane and forms
the proton channel
 Exchange metabolites at inner
mitochondrial membrane
- Exchange of anions against OH- ions and cations
against H+ ions → for transport of ionized metabolites
- Long chain fatty acids need carnitine system
- Symport pyruvate - H+
- Dicarboxylate and tricarboxylate anions require
specific carrier → linked to inorganic phosphate
(H2PO4-)
- Exchange ATP / ADP by adenine nucleotide
transporter
- Transport of oxaloacetate need transamination
process
 Oxidation of extramitochondrial NADH
- NADH cannot penetrate mitochondrial membrane
→ produced continuously in cytosol by 3
phosphoglyceraldehyde Dehidrogenase
- Aerobic conditions: not accumulated → be
oxidized by resp chain
- Transfer of reducing equivalents from cytosol to
mitochondrial require substrate pairs, linked by
suitable Dehidrogenase
 Oxidation of extramitochondrial NADH

- The mechanism:
1. Glycerophosphate shuttle → only 2 mol ATP
are formed per atom oxygen consumed → present
in brain, muscle, adipose, liver but deficient in
heart muscle
2. Malate shuttle → more universal utility →
more complex, due to the impermeability of
mitochondrial membrane to oxaloacetate
 OUTER INNER
MEMBRANE MEMBRANE

MITOCHONDRION
CYTOSOL

NAD+
Glycerol 3-phosphate Glycerol 3-phosphate
FAD
GLYCEROL-3- GLYCEROL-3-
PHOSPHATE PHOSPHATE
DEHYDROGENASE DEHYDROGENASE
(CYTOSOLIC) (MITHOCONDRIAL)

Dehydroxyacetone Dehydroxyacetone
NADH + phosphate FDH2
phosphate
H+

Respiratory Chain

Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the
mitochondrion
 INNER
MAMBRAN
CYTOSOL E MITHOCOND
RION
1
NAD+ Malate Malate NAD+

MALATE MALATE
DEHYDROGENASE DEHYDROGENASE
NADH NADH
+H+ Oxaloacetate -KG -KG Oxaloacetate +H+

TRANSAMI TRANSAMIN
NASE ASE
Glutamate Asp Asp Glutamate

H+ H+

Malate shuttle for transfer of reducing equivalents from the cytosol into the mitocondrion. 1.
Ketoglutarate transporter, 2. glutamate-aspartate transporter (note the proton symport with
glutamate)
 Inhibitor of electron transport and oxidative
phosphorilation
1. Agents that act as inhibitors of the electron
transport
→if there is block at any point in the electron
transport chain, all carries before the block will
accumulate in their reduce state, whereas those
after the block will accumulate in their oxidized
states . → Oxygen will not be comsumed, ATP will
not generated and TCA cycle will slow down
owing to the accumulation of NADH
→Blocking electrons transfer from Fe-S to
co Q , ie: barbiturates , pierisidin-A ,
rotenon , carboxine
succinate D’ase competitive inhibitor:
malonate
→ Blocking electrons transfer from cty b to
cyt c, ie: dimercaprol , antimycin A
→ Inhibitors of cytochrome oxidase: H2S ,
CO and CN
• 2. Inhibitors of phosphorylation (oligomycin,
atractyloside)
• 3. Un-couplers (dissociate oxidation from
phosphorylation)
Example : Dicyclohexylcarbodiimide (DCCD),
dinitrophenol, dinitrochressol,
pentachlorophenol,
chlorocarbonylcyanidephenilhydrazon (cccp)
 Uncoupling proteins (UCP)

• H+ transported into matrix by routes other than

ATP synthase and inner membrane transporters
• Much of BMR, mainly due to UCP
• These carrier proteins create a “proton leak” →
allow protons to re-enter the mitochondrial matrix
without energy being captured as ATP
• The energy is released as heat (thermogenesis)
• UCP-1 (thermogenin), provides body heat,
exclusively found in brown fat tissue
Uncoupling proteins (UCP)
• Brown fat (high content of mitochondria), unlike
the more abundant white fat, uses almost 90% of
its respiratory energy for thermogenesis in
response to cold in the neonate, and during arousal
in hibernating animals.
• In human, it gradually diminished
• Four additional UCP expressed by the human
genome (UCP-2 expressed ubiquitously, UCP-3
mainly in skeletal muscle, UCP-4 & UCP-5 in
brain) → physiologic function not well understood
• Could be of profound significance in
understanding of health issues as diabetes, obesity,
ca, thyroid desease and aging
• There is strong evidence that obesity induces
synthesis of UCP-2 in β-cells pancreas → β-cells
dysfunction in type 2 diabetes (ATP ↓)
• T3 (thyroxin hormone) stimulate thermogenesis in
rats by promoting synthesis UCP-3 in skeletal
muscle
• Common fever induced by infectious organisms
probably also due to UCP
 Creatine phosphate shuttle

• Facilitating transport of high energy phosphat

from mitochondria in active tissues
• Isoenzyme of creatine kinase (CKM), in
intermembrane space → catalyzing transfer ~ P
(ATP) to creatine:
~ P(ATP) + creatine → creatine-P , transported
into cytosol via protein pores → available for
generation of
extramitochondrial ATP
 H

P
N CREATINE H2N
KINASE

C NH C NH

N H3C N

ΔGO’ = 12.6 kJ/mol

COO- COO-

Creatine
Creatine
phosphate